Glycans play multi-faceted roles in many biological processes and aberrant glycosylation is associated with most of the diseases that affect mankind. Glycans are post-translation modifications of proteins that are involved in cell growth, cytokinesis, differentiation, transcription regulation, signal transduction, ligand-receptor binding, interactions of cells with other cells and extracellular matrix (ECM) and bacterial and viral infection, among other functions (see FIG. 1). Glycan misregulations and structural changes occur in most of the diseases that affect the human.
Lectin histochemistry methods are commonly used to stain tissue glycans on formalin-fixed paraffin-embedded (FFPE) sections (see FIG. 2). Some lectins have high affinities for the epitopes of certain glycans. For example, Concanavilin A (ConA) can be used as a ligand for high-mannose glycans, Sambucus nigra agglutinin (SNA) for sialylated glycans, and Alueria aurantia lectin (AAL) for fucosylated structures. Despite the impact the lectin chemistry has had on the field, it has limitations. For example, lectins provide minimal structural information about the stained epitopes, they are limited to one epitope at a time on each tissue section, they are quantitative and, compared with antibodies, have lower affinities for the glycans. Further, very few monoclonal antibodies have been developed for glycans.
Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) serves as a major technique for fast and accurate analysis of a number of molecules from complex mixtures such as cells, tissues and serum samples. Although MALDI-TOF MS has been successfully applied for detection, identification and validation of many peptides and molecules, it has proven ineffective for analyzing low abundance molecules from complex mixtures. Considering the extremely wide range of protein concentrations in plasma (i.e. from albumin at 1010 pg/mL to interleukins at 10 pg/mL), the lower-abundant proteins or peptides are dominated by the abundant serum contents and fail to be detected in a mixture. In addition, background and chemical noise (coming from desorbed matrix cluster) interfere with the MS signal and further compromise the sensitivity and detectability for low-abundance analytes. Despite the technological advances in MALDI-TOF instrumentation, the suboptimal transmission efficiency of the mass analyzer, and detection efficiency of the detector, also result in some loss of analyte, which is another factor that reduces the detection limit and sensitivity of MALDI-TOF MS with low-abundant analytes. On the other hand, the concentration of potential disease biomarkers, such as glycans lies in the lower range of concentrations in serum, particularly at the early stages of the disease where screening is crucial.
Therefore, there still exists a need to improve methods for generating structural information of glycans in FFPE sections, as well as the need to improve the sensitivity and detection limits of MALDI-TOF MS analytical methods for glycans, peptides and other target analytes, in order to be effective for biomarker discovery research.